Background In the vertebrate retina, six major types of neurons and Muller glia originate from common pools progenitors. Extensive investigations have revealed the importance of extracellular signals and intrinsic regulatory mechanisms that dictate cell-type specification in the neural retina. Our studies have yielded crucial insights into transcriptional regulation of rod and cone photoreceptor development (3). However, molecular pathways that dictate the differentiation of retinal neurons are still poorly understood. Results 1. Gene regulatory networks (GRN) in photoreceptor differentiation We have published the first detailed analysis of retinal transcriptome generated by next generation sequencing (NGS) technology (RNA-Seq). We propose RNA-Seq as a comprehensive and accurate method for quantitative/qualitative evaluation of mRNA content within a cell or tissue, and report our optimized protocols for low mRNA amounts (15 ng total RNA) and data analysis workflows as a framework for comparative investigations of expression profiles (1-2). Photoreceptor maturation requires several days (in mice) to weeks (in humans). Expression of NRL and CRX are not sufficient to activate phototransduction genes (including rhodopsin) and outer segment biogenesis, and additional downstream signals must be available. We are combining previously generated global expression profiles by Affymetrix GeneChips with global expression profiles by RNA-Seq of retina and flow-sorted mouse rod photoreceptors from 17 different stages, from embryonic day (E)11 to 24 months to reveal the regulatory network/pathways underlying photoreceptor maturation and homeostasis. To determine the downstream transcriptional cascade leading to functional rods, we have identified genome-wide targets of NRL by chromatin immuno-precipitation (ChIP) followed by NGS (ChIP-Seq) (4). We ascertained 300+ direct NRL target genes, of which 22 are associated with human retinal disease and 95 map to the region of disease loci. In vivo knockdown of a selection of NRL target genes results in abnormal morphology or death of rod photoreceptors. Finally, we have discovered that KDM5B and MEF2C are new secondary nodes downstream of NRL in the rod GRN. In particular, MEF2C protein is implicated in rhodopsin gene regulation, suggesting its involvement in rod photoreceptor cell homeostasis (5). Stringent control of rhodopsin expression is critical for photoreceptor maintenance and survival. Rhodopsin distal enhancer region (RER) is required for quantitative precise rod-specific expression. We have isolated several RER-bound proteins from bovine retinal extract and analyzed them by mass spectrometry (MS); of these, the most number of unique peptides were obtained for non-POU domain octamer-binding protein (NonO/p54nrb) that is associated with both splicing and transcription. We further sought to identify the components of the multiprotein NRL-containing transcriptional complex(es) bound to rhodopsin promoter. We have isolated, from adult bovine retina, an approximately 500 KDa complex that contains NRL, CRX and NR2E3. Immunoblotting and MS analysis will help us define all components of this complex. In parallel, we are using yeast-two-hybrid screening of developing and adult retina libraries to define NRL-interactome. 2. Epigenetic regulation in retinal development Epigenetic modulators contribute to the complexity of gene regulation. DNA methyltransferases (Dnmts) play fundamental roles in development but their contributions to retinogenesis have not been delineated. Our recent data indicate that Dnmt1 knockdown in RPE (but not in neural retina) during early retinal development leads to abnormal RPE morphology and aberrant photoreceptor outer segment morphogenesis. To determine how chromatin modifications control gene expression, we are generating global methylome profiles of photoreceptors during differentiation. Furthermore, we have established a protocol to examine histone modifications using 25-100K purified rod photoreceptors. We are using histone H3K4me3 and H3K27me3 marks (active and silent promoters, respectively) and RNA polymerase II occupancy to investigate epigenetic control of rhodopsin and other promoters for photoreceptor-specific genes. Epigenetic data will be superimposed on expression profiles and transcription factor ChIP-Seq data for an integrated analysis of GRNs. 3. Regulation of NRL function We have previously described modulation of NRL stability and activity by post-translational modifications (PTMs), including phosphorylation and SUMOylation. Our studies suggest that glycogen synthase kinase 3 (GSK3), casein kinase 2 (CKII), and a candidate E3-sumo ligase, protein inhibitor of activated STAT, 3 (PIAS3) are involved in regulating NRL (7). We now have found that phosphorylation at S50 seems to trigger successive phosphorylations at S46, T42 and S38 by GSK3, leading to NRL degradation by the proteasome. Conversely, SUMOylation and mutations of serine residues enhance NRL protein stability. 4. Establishment of photoreceptor cell polarity and ciliogenesis Planar cell polarity (PCP)-associated Prickle genes (Pk1 and Pk2) are tissue polarity genes necessary for the establishment of PCP in Drosophila. By in vivo knockdown of Pk1 in neonatal mouse retina we have identified defects in outer segments and axon terminals of photoreceptors. These data suggest a role for Pk1 in establishing the conditions for PCP in photoreceptors. BBS9/PTHB1 is one of the seven core proteins that form a stable complex called BBSome, implicated in trafficking of proteins to primary cilia, and is among fifteen known BBS-associated genes. Upon knockdown of BBS9 in zebrafish, we detected developmental abnormalities in retina and reduced number and length of cilia in Kupffers vesicle, suggesting a key role of BBS9 in cilia biogenesis (8). 5. Synaptogenesis Formation of specific synapses with bipolar and horizontal cells is a fundamental step in maturation of photoreceptors. Our recent data using cone-only Nrl-/- retina and rod-only Crxp-Nrl retina show that synaptic terminals in Nrl-/- retina switch from spherule to pedicle-like morphology and that bipolar cell dendritic arbors are altered. Thus, NRL seems to control the expression of genes involved in morphogenesis of presynaptic assembly and synapse formation in developing mouse retina. By comparing temporal rod gene profiles of wild type vs Nrl-/- retina, we have identified 111 candidate genes; of these, ten have been selected for in vivo knockdown analysis to explore their role in axonogenesis and synapse formation. 6. Signaling pathways during retinogenesis All major signaling pathways, including Notch, Hedgehog and Wnt, contribute to retinal development. We focused on two Frizzled receptors, Fz5 and Fz8, based on previous work showing retinal developmental defects in mouse mutants. Compound Fz5/Fz8 mutants reveal a dose-dependent regulation of signaling by Fz5 and Fz8 in optic fissure/disc formation and in progenitor pool expansion (6). We propose that non-canonical Frizzled signaling affects neuroblast organization and apical-basal polarity in retinal neurogenesis. Significance Our studies provide new insights into photoreceptor biology and disease. We are defining the role of specific genes and epigenetic signals in establishing photoreceptor function during retinal development. This project complements our efforts to engineer stem cells into photoreceptors and functionally integrate them into the disrupted architecture of degenerating retinas after transplantation (see EY000474) and will assist in developing novel paradigms for treatment of retinal and macular degenerative diseases.